Project Summary Natural products are an essential source of inspiration for medicines. The World Health Organization estimates that ~80% of the world's population relies on traditional medicines made from natural products (ref). The Pharmaceutical industry has ~50% of all drugs based on or derived from natural products. Unfortunately, the synthesis of these molecules is often prohibitively complex, requiring the installation of multiple functional groups with very specific 3D architecture critical to their biological activity. Oxidative dearomatization of phenolic compounds is a powerful transformation for the synthesis of complex molecules, as it introduces stereochemistry and generates products primed for further reaction. For example, this reaction is the key step in the biosynthesis of isochromophilone II and luteusin A, inhibitors of the interaction between gp120, a glycoprotein found on the surface of HIV, and CD4, on the surface of T-cells central to the HIV invasion mechanism. Inhibition of this protein-protein interaction is hypothesized to disrupt the entry of the human immunodeficiency virus (HIV) into cells. Only a limited number of enantioselective methods have been reported for oxidative dearomatization, limiting the application of this transformation in synthetic chemistry. To achieve high degrees of stereoselectivity, stoichiometric amounts of the chiral metal complexes are required in addition to lengthy reaction times, cryogenic temperatures, and harsh conditions. Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions and the use of environmentally benign reagents. These advantages have created a demand for new biocatalysts that expand the portfolio of complexity-generating reactions available to synthetic chemists. However, the tradeoff that often exists between the substrate scope of a biocatalyst and its selectivity limits the application of enzymes in synthesis. On this proposal, we explore a panel of FAD-monooxygenases, TropB, AfoD, AzaH, and SorbC containing complementary substrates scopes and high levels of site- and stereoselectivity across a range of structurally diverse substrates. This research proposal aims to aim to develop a suite of catalysts with complementary selectivities in order to provide an efficient route to valuable chiral intermediates for the synthesis of bioactive molecules. This research will focus on FAF-monooxygenases, TropB, AfoD, AzaH, and SorbC. Were I will (1) Determine the binding of the panel of enzymes (2) Transpose this information to expand the substrate scope of TropB by modification of residues utilizing protein engineering (3) Further enhance the reactivity of the biocatalyst by utilizing C8-FAD analogs. Such tools will provide an efficient route to valuable chiral intermediates for the synthesis of bioactive molecules.